Nucleotide sequences coding for the MtrA and/or MtrB proteins

ABSTRACT

The present invention provides nucleotide sequences from Coryneform bacteria which code for the MtrA and/or MtrB proteins and processes for the fermentative preparation of amino acids using bacteria in which the mtrA and/or mtrB genes are attenuated.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides nucleotide sequences from Coryneformbacteria which code for the MtrA and/or MtrB proteins and processes forthe fermentative preparation of amino acids using bacteria in which themtrA and/or mtrB genes are attenuated.

2. Discussion of the Background

L-Amino acids, in particular L-lysine, are used in human medicine and inthe pharmaceuticals industry, in the foodstuffs industry and veryparticularly in animal nutrition.

It is known that amino acids are prepared by fermentation from strainsof Coryneform bacteria, in particular Corynebacterium glutamicum.Because of their great importance, work is constantly being undertakento improve the preparation processes. Improvements to the process canrelate to fermentation measures, such as, for example, stirring andsupply of oxygen, or the composition of the nutrient media, such as, forexample, the sugar concentration during the fermentation, or the workingup to the product form by, for example, ion exchange chromatography, orthe intrinsic output properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used toimprove the output properties of these microorganisms. Strains which areresistant to antimetabolites or are auxotrophic for metabolites ofregulatory importance and which produce amino acids are obtained in thismanner.

Methods of the recombinant DNA technique have also been employed forsome years for improving the strain of Corynebacterium strains whichproduce L-amino acid, by amplifying individual amino acid biosynthesisgenes and investigating the effect on the amino acid production.

However, there remains a critical need for improved methods of producingL-amino acids and thus for the provision of strains of bacteriaproducing higher amounts of L-amino acids. On a commercial or industrialscale even small improvements in the yield of L-amino acids, or theefficiency of their production, are economically significant. Prior tothe present invention, it was not recognized that attenuating the mtrAand/or mtrB genes encoding response regulator and histidine proteinkinase proteins, respectively, would improve L-amino acid yields.

SUMMARY OF THE INVENTION

An object of the present invention is to provide novel measures for theimproved production of L-amino acids or amino acid, where these aminoacids include L-asparagine, L-threonine, L-serine, L-glutamate,L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine,L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine,L-tryptophan, L-arginine and the salts (monohydrochloride or sulfate)thereof.

One object of the present invention is providing a novel process forimproving the fermentative production of said L-amino acids,particularly L-lysine. Such a process includes attenuated bacteria,preferably attenuated coryneform bacteria, which express attenuatedamounts MtrA and/or MtrB proteins or proteins that have responseregulator or histidine protein kinase activities.

Thus, another object of the present invention is providing such abacterium, which expresses an enhanced amount of the MtrA and/or MtrBproteins or gene products of the mtrA and/or mtrB genes.

Another object of the present invention is providing a bacterium,preferably a Coryneform bacterium, which expresses a polypeptide thathas an attenuated MtrA response regulator and/or histidine proteinkinase activities.

Another object of the invention is to provide a nucleotide sequenceencoding a polypeptide having the MtrA and/or MtrB sequences. Oneembodiment of such a sequence providing both mtrA and mtrB is thenucleotide sequence of SEQ ID NO: 1. Additionally, nucleotides 542 to1219 of SEQ ID NO:1 comprise the MtrA coding region and nucleotides 1310to 2818 comprise the MtrB coding region.

A further object of the invention is a method of making proteins orisolated polypeptides having MtrA response regulator and/or histidineprotein kinase activities, as well as use of such isolated polypeptidesin the production of amino acids. One embodiment of the MtrA polypeptideis the polypeptide having the amino acid sequence of SEQ ID NO: 2. Oneembodiment of the MtrB polypeptide is the polypeptide having the aminoacid sequence of SEQ ID NO:3.

Other objects of the invention include methods of detecting nucleic acidsequences homologous to SEQ ID NO: 1, particularly nucleic acidsequences encoding polypeptides that have MtrA response regulator and/orhistidine protein kinase activities, and methods of making nucleic acidsencoding such polypeptides.

The above objects highlight certain aspects of the invention. Additionalobjects, aspects and embodiments of the invention are found in thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Map of the plasmid pCR2.1mtrAint.

FIG. 2: Map of the plasmid pCR2.1mtrBint.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art of molecular biology. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed herein. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting.

Reference is made to standard textbooks of molecular biology thatcontain definitions and methods and means for carrying out basictechniques, encompassed by the present invention. See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1989), Current Protocols in MolecularBiology, Ausebel et al (eds), John Wiley and Sons, Inc. New York (2000)and the various references cited therein.

“L-amino acids” or “amino acids” as used herein mean one or more aminoacids, including their salts, chosen from the group consisting ofL-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine,L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine,L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine.L-Lysine is particularly preferred.

When L-lysine or lysine are mentioned in the following, not only thebases but also the salts, such as e.g. lysine monohydrochloride orlysine sulfate, are meant by this.

The invention provides an isolated polynucleotide from Coryneformbacteria, comprising a polynucleotide sequence which codes for the mtrAgene and/or the mtrB gene, chosen from the group consisting of

-   a) polynucleotide which is identical to the extent of at least 70%    to a polynucleotide which codes for a polypeptide which comprises    the amino acid sequence of SEQ ID No. 2,-   b) polynucleotide which is identical to the extent of at least 70%    to a polynucleotide which codes for a polypeptide which comprises    the amino acid sequence of SEQ ID No. 3,-   c) polynucleotide which codes for a polypeptide which comprises an    amino acid sequence which is identical to the extent of at least 70%    to the amino acid sequence of SEQ ID No. 2,-   d) polynucleotide which codes for a polypeptide which comprises an    amino acid sequence which is identical to the extent of at least 70%    to the amino acid sequence of SEQ ID No. 3,-   e) polynucleotide which is complementary to the polynucleotides of    a), b), c) or d), and-   f) polynucleotide comprising at least 15 successive nucleotides of    the polynucleotide sequence of a), b), c), d) or e),    the polypeptides preferably having the activity of the response    regulator MtrA and/or of the histidine protein kinase MtrB.

The invention also provides the above-mentioned polynucleotides, thesepreferably being DNAs which are capable of replication, comprising:

-   (i) the nucleotide sequence, in particular between positions 1 to    2717, shown in SEQ ID No.1, or-   (ii) at least one sequence which corresponds to sequence (i) within    the degeneracy of the genetic code, or-   (iii) at least one sequence which hybridizes with the sequences    complementary to sequences (i) or (ii) and optionally-   (iv) sense mutations of neutral function in (i) which do not modify    the activity of the protein/polypeptide.

Finally, the invention also provides polynucleotides chosen from thegroup consisting of

-   a) polynucleotides comprising at least 15 successive nucleotides    chosen from the nucleotide sequence of SEQ ID No. 1 between    positions 1 and 541,-   b) polynucleotides comprising at least 15 successive nucleotides    chosen from the nucleotide sequence of SEQ ID No. 1 between    positions 542 and 1219,-   c) polynucleotides comprising at least 15 successive nucleotides    chosen from the nucleotide sequence of SEQ ID No. 1 between    positions 1220 and 1309,-   d) polynucleotides comprising at least 15 successive nucleotides    chosen from the nucleotide sequence of SEQ ID No. 1 between    positions 1310 and 2717,-   e) polynucleotides comprising at least 15 successive nucleotides    chosen from the nucleotide sequence of SEQ ID No. 1 between    positions 2718 and 3910.

The invention also provides:

-   polynucleotides, in particular DNAs, which are capable of    replication and comprise the nucleotide sequence as shown in SEQ ID    No.1;-   polynucleotides which code for polypeptides which comprise the amino    acid sequence as shown in SEQ ID No. 2 or SEQ ID No.3;-   vectors containing parts of the polynucleotide according to the    invention, having at least 15 successive nucleotides of the sequence    claimed,-   and Coryneform bacteria in which the mtrA gene and/or the mtrB gene    is attenuated, in particular by an insertion or deletion.

The invention also provides polynucleotides, which substantiallycomprise a polynucleotide sequence, which are obtainable by screening bymeans of hybridization of a corresponding gene library of a Coryneformbacterium, which comprises the complete gene or parts thereof, with aprobe which comprises the sequence of the polynucleotide according tothe invention according to SEQ ID No.1 or a fragment thereof, andisolation of the polynucleotide sequence mentioned.

Polynucleotides which comprise the sequences according to the inventionare suitable as hybridization probes for RNA, cDNA and DNA, in order toisolate, in the full length, nucleic acids or polynucleotides or geneswhich code for the response regulator MtrA and/or the histidine proteinkinase MtrB, or to isolate those nucleic acids or polynucleotides orgenes which have a high similarity with the sequence of the mtrA geneand/or the mtrB gene. Additionally, methods employing DNA chips,microarrays or similar recombinant DNA technology that enables highthroughput screening of DNA and polynucleotides which encode theglyceraldehydes 3-phosphate dehydrogenase 2 protein or polynucleotideswith homology to the gap2 gene as described herein. Such methods areknown in the art and are described, for example, in Current Protocols inMolecular Biology, Ausebel et al (eds), John Wiley and Sons, Inc. NewYork (2000).

Polynucleotides which comprise the sequences according to the inventionare furthermore suitable as primers with the aid of which DNA of geneswhich code for the response regulator MtrA and/or the histidine proteinkinase MtrB can be prepared by the polymerase chain reaction (PCR).

Such oligonucleotides which serve as probes or primers comprise at least25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24, veryparticularly preferably at least 15, 16, 17, 18 or 19 successivenucleotides. Oligonucleotides with a length of at least 31, 32, 33, 34,35, 36, 37, 38, 39 or 40 or at least 41, 42, 43, 44, 45, 46, 47, 48, 49or 50 nucleotides are also suitable. Oligonucleotides with a length ofat least 100, 150, 200, 250 or 300 nucleotides are optionally alsosuitable.

“Isolated” means separated out of its natural environment.

“Polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

The polynucleotides according to the invention include a polynucleotideaccording to SEQ ID No. 1 or a fragment prepared therefrom and alsothose which are at least 70% to 80%, preferably at least 81% to 85%,particularly preferably at least 86% to 90% and very particularlypreferably at least 91%, 93%, 95%, 97% or 99% identical to thepolynucleotide according to SEQ ID No. 1 or a fragment preparedtherefrom.

“Polypeptides” are understood as meaning peptides or proteins whichcomprise two or more amino acids bonded via peptide bonds.

The polypeptides according to the invention include a polypeptideaccording to SEQ ID No. 2 and SEQ ID No. 3, in particular those with thebiological activity of the response regulator MtrA and the histidineprotein kinase MtrB, and also those which are at least 70% to 80%,preferably at least 81% to 85%, particularly preferably at least 86% to90% and very particularly preferably at least 91%, 93%, 95%, 97% or 99%identical to the polypeptide according to SEQ ID No. 2 and SEQ ID No. 3and have the activities mentioned.

The invention furthermore relates to a process for the fermentativepreparation of amino acids chosen from the group consisting ofL-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine,L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine,L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginineusing Coryneform bacteria which in particular already produce aminoacids and in which the nucleotide sequences which code for the mtrAand/or mtrB gene are attenuated, in particular eliminated or expressedat a low level.

The term “attenuation” in this connection describes the reduction orelimination of the intracellular activity of one or more enzymes(proteins) in a microorganism which are coded by the corresponding DNA,for example by using a weak promoter or using a gene or allele whichcodes for a corresponding enzyme with a low activity or inactivates thecorresponding gene or enzyme (protein), and optionally combining thesemeasures.

By attenuation measures, the activity or concentration of thecorresponding protein is in general reduced to 0 to 75%, 0 to 50%, 0 to25%, 0 to 10% or 0 to 5% of the activity or concentration of thewild-type protein or of the activity or concentration of the protein inthe starting microorganism.

Preferably, a bacterial strain with attenuated expression of the mtrAand/or mtrB genes encoding a mtrA response regulator or histidineprotein kinase will improve amino acid yield at least 1%.

The microorganisms provided by the present invention can prepare aminoacids from glucose, sucrose, lactose, fructose, maltose, molasses,starch, cellulose or from glycerol and ethanol. They can berepresentatives of Coryneform bacteria, in particular of the genusCorynebacterium. Of the genus Corynebacterium, there may be mentioned inparticular the species Corynebacterium glutamicum, which is known amongexperts for its ability to produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular of thespecies Corynebacterium glutamicum (C. glutamicum), are in particularthe known wild-type strains

-   -   Corynebacterium glutamicum ATCC13032    -   Corynebacterium acetoglutamicum ATCC15806    -   Corynebacterium acetoacidophilum ATCC13870    -   Corynebacterium melassecola ATCC17965    -   Corynebacterium thermoaminogenes FERM BP-1539    -   Brevibacterium flavum ATCC14067    -   Brevibacterium lactofermentum ATCC13869 and    -   Brevibacterium divaricatum ATCC14020        and L-amino acid-producing mutants or strains prepared        therefrom.

The new genes mtrA and mtrB gene of C. glutamicum which code for theresponse regulator MtrA and the histidine protein kinase MtrB have beenisolated. The two proteins are part of a two-component system.Two-component regulation systems are distinguished in that variousresponse regulator proteins can be activated by sensor kinases.

To isolate the mtrA gene, the mtrB gene or also other genes of C.glutamicum, a gene library of this microorganism is first set up inEscherichia coli (E. coli). The setting up of gene libraries isdescribed in generally known textbooks and handbooks. The textbook byWinnacker: Gene und Klone, Eine Einfuhrung in die Gentechnologie (VerlagChemie, Weinheim, Germany, 1990), or the handbook by Sambrook et al.:Molecular Cloning, A Laboratory Manual (Cold Spring Harbor LaboratoryPress, 1989) may be mentioned as an example. A well-known gene libraryis that of the E. coli K-12 strain W3110 set up in λ vectors by Koharaet al. (Cell 50, 495-508 (1987)). Bathe et al. (Molecular and GeneralGenetics, 252:255-265, 1996) describe a gene library of C. glutamicumATCC13032, which was set up with the aid of the cosmid vector SuperCos I(Wahl et al., 1987, Proceedings of the National Academy of Sciences USA,84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988,Nucleic Acids Research 16:1563-1575). Börmann et al. (MolecularMicrobiology 6(3), 317-326)) (1992)) in turn describe a gene library ofC. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, 1980,Gene 11, 291-298).

To prepare a gene library of C. glutamicum in E. coli it is alsopossible to use plasmids such as pBR322 (Bolivar, 1979, Life Sciences,25, 807-818) or pUC9 (Vieira et al., 1982, Gene, 19:259-268). Suitablehosts are, in particular, those E. coli strains which are restriction-and recombination-defective, such as, for example, the strain DH5αmcr,which has been described by Grant et al. (Proceedings of the NationalAcademy of Sciences USA, 87 (1990) 4645-4649). The long DNA fragmentscloned with the aid of cosmids or other λ vectors can then in turn besubcloned and subsequently sequenced in the usual vectors which aresuitable for DNA sequencing, such as is described e.g. by Sanger et al.(Proceedings of the National Academy of Sciences of the United States ofAmerica, 74:5463-5467, 1977).

The resulting DNA sequences can then be investigated with knownalgorithms or sequence analysis programs, such as e.g. that of Staden(Nucleic Acids Research 14, 217-232(1986)), that of Marck (Nucleic AcidsResearch 16, 1829-1836 (1988)) or the GCG program of Butler (Methods ofBiochemical Analysis 39, 74-97 (1998)).

The new DNA sequences of C. glutamicum which code for the mtrA and mtrBgenes and which, as SEQ ID No. 1, are a constituent of the presentinvention have been found. The amino acid sequence of the correspondingprotein has furthermore been derived from the present DNA sequence bythe methods described above. The resulting amino acid sequences of themtrA and mtrB gene products are shown in SEQ ID No. 2 and SEQ ID No. 3.It is known that enzymes endogenous in the host can split off theN-terminal amino acid methionine or formylmethionine of the proteinformed.

Coding DNA sequences which result from SEQ ID No. 1 by the degeneracy ofthe genetic code are also a constituent of the invention. In the sameway, DNA sequences which hybridize with SEQ ID No. 1 or parts of SEQ IDNo. 1 are a constituent of the invention. Conservative amino acidexchanges, such as e.g. exchange of glycine for alanine or of asparticacid for glutamic acid in proteins, are furthermore known among expertsas “sense mutations” which do not lead to a fundamental change in theactivity of the protein, i.e. are of neutral function. Such mutationsare also called, inter alia, neutral substitutions. It is furthermoreknown that changes on the N and/or C terminus of a protein cannotsubstantially impair or can even stabilize the function thereof.Information in this context can be found by the expert, inter alia, inBen-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), inO'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (ProteinSciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology6:1321-1325 (1988)) and in known textbooks of genetics and molecularbiology. Amino acid sequences which result in a corresponding mannerfrom SEQ ID No. 2 are also a constituent of the invention.

In the same way, DNA sequences which hybridize with SEQ ID No. 1 orparts of SEQ ID No. 1 are a constituent of the invention. Finally, DNAsequences which are prepared by the polymerase chain reaction (PCR)using primers which result from SEQ ID No. 1 are a constituent of theinvention. Such oligonucleotides typically have a length of at least 15nucleotides.

Instructions for identifying DNA sequences by means of hybridization canbe found by the expert, inter alia, in the handbook “The DIG SystemUsers Guide for Filter Hybridization” from Boehringer Mannheim GmbH(Mannheim, Germany, 1993) and in Liebl et al. (International Journal ofSystematic Bacteriology 41: 255-260 (1991)). The hybridization takesplace under stringent conditions, that is to say only hybrids in whichthe probe and target sequence, i.e. the polynucleotides treated with theprobe, are at least 70% identical are formed. It is known that thestringency of the hybridization, including the washing steps, isinfluenced or determined by varying the buffer composition, thetemperature and the salt concentration. The hybridization reaction ispreferably carried out under a relatively low stringency compared withthe washing steps (Hybaid Hybridisation Guide, Hybaid Limited,Teddington, UK, 1996).

A 5×SSC buffer at a temperature of approx. 50° C.-68° C., for example,can be employed for the hybridization reaction. Probes can alsohybridize here with polynucleotides which are less than 70% identical tothe sequence of the probe. Such hybrids are less stable and are removedby washing under stringent conditions. This can be achieved, forexample, by lowering the salt concentration to 2×SSC and optionallysubsequently 0.5×SSC (The DIG System User's Guide for FilterHybridisation, Boehringer Mannheim, Mannheim, Germany, 1995) atemperature of approx. 50° C.-68° C. being established. It is optionallypossible to lower the salt concentration to 0.1×SSC. Polynucleotidefragments which are, for example, at least 70% or at least 80% or atleast 90% to 95% or at least 96% to 99% identical to the sequence of theprobe employed can be isolated by increasing the hybridizationtemperature stepwise from 50° C. to 68° C. in steps of approx. 1-2° C.It is also possible to isolate polynucleotide fragments which arecompletely identical to the sequence of the probe employed. Furtherinstructions on hybridization are obtainable on the market in the formof so-called kits (e.g. DIG Easy Hyb from Roche Diagnostics GmbH,Mannheim, Germany, Catalogue No. 1603558).

Instructions for amplification of DNA sequences with the aid of thepolymerase chain reaction (PCR) can be found by the expert, inter alia,in the handbook by Gait: oligonucleotide Synthesis: A Practical Approach(IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (SpektrumAkademischer Verlag, Heidelberg, Germany, 1994).

It has been found that Coryneform bacteria produce amino acids in animproved manner after attenuation of the mtrA gene and/or the mtrB gene.

To achieve an attenuation, either the expression of the mtrA gene and/orof the mtrB gene or the catalytic or regulatory properties of the enzymeproteins can be reduced or eliminated. The two measures can optionallybe combined.

The reduction in gene expression can take place by suitable culturing orby genetic modification (mutation) of the signal structures of geneexpression. Signal structures of gene expression are, for example,repressor genes, activator genes, operators, promoters, attenuators,ribosome binding sites, the start codon and terminators. The expert canfind information on this e.g. in the patent application WO 96/15246, inBoyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuiland Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen andHammer (Biotechnology and Bioengineering 58: 191 (1998)), in Patek etal. (Microbiology 142: 1297 (1996)), Vasicova et al. (Journal ofBacteriology 181: 6188 (1999)) and in known textbooks of genetics andmolecular biology, such as e.g. the textbook by Knippers (“MolekulareGenetik”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) orthat by Winnacker (“Gene und Klone”, VCH Verlagsgesellschaft, Weinheim,Germany, 1990).

Mutations which lead to a change or reduction in the catalyticproperties of enzyme proteins are known from the prior art; exampleswhich may be mentioned are the works by Qiu and Goodman (Journal ofBiological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (BioscienceBiotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (“DieThreonindehydratase aus Corynebacterium glutamicum: Aufhebung derallosterischen Regulation und Struktur des Enzyms”, Reports from theJülich Research Center, Jül-2906, ISSN09442952, Jülich, Germany, 1994).Summarizing descriptions can be found in known textbooks of genetics andmolecular biology, such as e.g. that by Hagemann (“Allgemeine Genetik”,Gustav Fischer Verlag, Stuttgart, 1986).

Possible mutations are transitions, transversions, insertions anddeletions. Depending on the effect of the amino acid exchange on theenzyme activity, “missense mutations” or “nonsense mutations” arereferred to. Insertions or deletions of at least one base pair (bp) in agene lead to frame shift mutations, as a consequence of which incorrectamino acids are incorporated or translation is interrupted prematurely.Deletions of several codons typically lead to a complete loss of theenzyme activity. Instructions on generation of such mutations are priorart and can be found in known textbooks of genetics and molecularbiology, such as e.g. the textbook by Knippers (“Molekulare Genetik”,6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that byWinnacker (“Gene und Klone”, VCH Verlagsgesellschaft, Weinheim, Germany,1990) or that by Hagemann (“Allgemeine Genetik”, Gustav Fischer Verlag,Stuttgart, 1986).

A common method of mutating genes of C. glutamicum is the method of“gene disruption” and “gene replacement” described by Schwarzer andPuhler (Bio/Technology 9, 84-87 (1991)).

In the method of gene disruption a central part of the coding region ofthe gene of interest is cloned in a plasmid vector which can replicatein a host (typically E. coli), but not in C. glutamicum. Possiblevectors are, for example, pSUP301 (Simon et al., Bio/Technology 1,784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73(1994)), pK18mobsacB or pK19mobsacB (Jäger et al., Journal ofBacteriology 174: 5462-65 (1992)), pGEM-T (Promega Corporation, Madison,Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry269:32678-84; U.S. Pat. No. 5,487,993), pCR®Blunt (Invitrogen,Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234:534-541 (1993)) or pEMI (Schrumpf et al, 1991, Journal of Bacteriology173:4510-4516). The plasmid vector which contains the central part ofthe coding region of the gene is then transferred into the desiredstrain of C. glutamicum by conjugation or transformation. The method ofconjugation is described, for example, by Schafer et al. (Applied andEnvironmental Microbiology 60, 756-759′ (1994)). Methods fortransformation are described, for example, by Thierbach et al. (AppliedMicrobiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan(Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)). After homologousrecombination by means of a “cross-over” event, the coding region of thegene in question is interrupted by the vector sequence and twoincomplete alleles are obtained, one lacking the 3′ end and one lackingthe 5′ end. This method has been used, for example, by Fitzpatrick etal. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) toeliminate the recA gene of C. glutamicum.

In the method of “gene replacement”, a mutation, such as e.g. adeletion, insertion or base exchange, is established in vitro in thegene of interest. The allele prepared is in turn cloned in a vectorwhich is not replicative for C. glutamicum and this is then transferredinto the desired host of C. glutamicum by transformation or conjugation.After homologous recombination by means of a first “cross-over” eventwhich effects integration and a suitable second “cross-over” event whicheffects excision in the target gene or in the target sequence, theincorporation of the mutation or of the allele is achieved. This methodwas used, for example, by Peters-Wendisch et al. (Microbiology 144,915-927 (1998)) to eliminate the pyc gene of C. glutamicum by adeletion.

A deletion, insertion or a base exchange can be incorporated into themtrA gene and/or the mtrB gene in this manner.

In addition, it may be advantageous for the production of L-amino acidsto enhance, in particular over-express, one or more enzymes of theparticular biosynthesis pathway, of glycolysis, of anaplerosis, of thecitric acid cycle, of the pentose phosphate cycle, of amino acid exportand optionally regulatory proteins, in addition to the attenuation ofthe mtrA gene and/or mtrB gene.

The term “enhancement” in this connection describes the increase in theintracellular activity of one or more enzymes (proteins) in amicroorganism which are coded by the corresponding DNA, for example byincreasing the number of copies of the gene or genes, using a potentpromoter or using a gene or allele which codes for a correspondingenzyme (protein) having a high activity, and optionally combining thesemeasures.

By enhancement measures, in particular over-expression, the activity orconcentration of the corresponding protein is in general increased by atleast 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to amaximum of 1000% or 2000%, based on that of the wild-type protein or theactivity or concentration of the protein in the starting microorganism.

Thus, for the preparation of L-amino acids, in addition to theattenuation of the mtrA gene and/or the mtrB gene at the same time oneor more of the genes chosen from the group consisting of

-   -   the dapA gene which codes for dihydrodipicolinate synthase (EP-B        0 197 335),    -   the gap gene which codes for glyceraldehyde 3-phosphate        dehydrogenase (Eikmanns (1992), Journal of Bacteriology        174:6076-6086),    -   the tpi gene which codes for triose phosphate isomerase        (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),    -   the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns        (1992), Journal of Bacteriology 174:6076-6086),    -   the zwf gene which codes for glucose 6-phosphate dehydrogenase        (JP-A-09224661),    -   the pyc gene which codes for pyruvate carboxylase (DE-A-198 31        609),    -   the mqo gene which codes for malate-quinone oxidoreductase        (Molenaar et al., European Journal of Biochemistry 254, 395-403        (1998)),    -   the lysC gene which codes for a feed-back resistant aspartate        kinase (Accession No.P26512; EP-B-0387527; EP-A-0699759; WO        00/63388),    -   the lysE gene which codes for lysine export (DE-A-195 48 222),    -   the hom gene which codes for homoserine dehydrogenase (EP-A        0131171),    -   the ilvA gene which codes for threonine dehydratase (Möckel et        al., Journal of Bacteriology (1992) 8065-8072)) or the ilvA(Fbr)        allele which codes for a “feed back resistant” threonine        dehydratase (Möckel et al., (1994) Molecular Microbiology 13:        833-842),    -   the ilvBN gene which codes for acetohydroxy-acid synthase (EP-B        0356739),    -   the ilvD gene which codes for dihydroxy-acid dehydratase (Sahm        and Eggeling (1999) Applied and Environmental Microbiology 65:        1973-1979),    -   the zwa1 gene which codes for the Zwa1 protein (DE: 19959328.0,        DSM 13115)        can be enhanced, in particular over-expressed.

It may furthermore be advantageous for the production of amino acids, inaddition to the attenuation of the mtrA gene and/or the mtrB gene, atthe same time for one or more of the genes chosen from the groupconsisting of

-   -   the pck gene which codes for phosphoenol pyruvate carboxykinase        (DE 199 50 409.1, DSM 13047),    -   the pgi gene which codes for glucose 6-phosphate isomerase (U.S.        Ser. No. 09/396,478, DSM 12969),    -   the poxB gene which codes for pyruvate oxidase (DE:1995 1975.7,        DSM 13114),    -   the zwa2 gene which codes for the Zwa2 protein (DE: 19959327.2,        DSM 13113)        to be attenuated, in particular for the expression thereof to be        reduced.

In addition to the attenuation of the mtrA gene and/or the mtrB gene itmay furthermore be advantageous for the production of amino acids toeliminate undesirable side reactions (Nakayama: “Breeding of Amino AcidProducing Microorganisms”, in: Overproduction of Microbial Products,Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The invention also provides the microorganisms prepared according to theinvention, and these can be cultured continuously or discontinuously inthe batch process (batch culture) or in the fed batch (feed process) orrepeated fed batch process (repetitive feed process) for the purpose ofproduction of L-amino acids. A summary of known culture methods isdescribed in the textbook by Chmiel (Bioprozesstechnik 1. Einfuhrung indie Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren und periphere Einrichtungen(Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose,fructose, maltose, molasses, starch and cellulose, oils and fats, suchas, for example, soya oil, sunflower oil, groundnut oil and coconut fat,fatty acids, such as, for example, palmitic acid, stearic acid andlinoleic acid, alcohols, such as, for example, glycerol and ethanol, andorganic acids, such as, for example, acetic acid, can be used as thesource of carbon. These substances can be used individually or as amixture.

Organic nitrogen-containing compounds, such as peptones, yeast extract,meat extract, malt extract, corn steep liquor, soya bean flour and urea,or inorganic compounds, such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate, can be usedas the source of nitrogen. The sources of nitrogen can be usedindividually or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used asthe source of phosphorus. The culture medium must furthermore comprisesalts of metals, such as, for example, magnesium sulfate or ironsulfate, which are necessary for growth. Finally, essential growthsubstances, such as amino acids and vitamins, can be employed inaddition to the above-mentioned substances. Suitable precursors canmoreover be added to the culture medium. The starting substancesmentioned can be added to the culture in the form of a single batch, orcan be fed in during the culture in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammoniaor aqueous ammonia, or acid compounds, such as phosphoric acid orsulfuric acid, can be employed in a suitable manner to control the pH ofthe culture. Antifoams, such as, for example, fatty acid polyglycolesters, can be employed to control the development of foam. Suitablesubstances having a selective action, such as, for example, antibiotics,can be added to the medium to maintain the stability of plasmids. Tomaintain aerobic conditions, oxygen or oxygen-containing gas mixtures,such as, for example, air, are introduced into the culture. Thetemperature of the culture is usually 20° C. to 45° C., and preferably25° C. to 40° C. Culturing is continued until a maximum of the desiredproduct has formed. This target is usually reached within 10 hours to160 hours.

Methods for the determination of L-amino acids are known from the priorart. The analysis can thus be carried out, for example, as described bySpackman et al. (Analytical Chemistry, 30, (1958), 1190) by anionexchange chromatography with subsequent ninhydrin derivation, or it canbe carried out by reversed phase HPLC, for example as described byLindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).

The process according to the invention is used for fermentativepreparation of amino acids.

The following microorganisms were deposited as a pure culture on 11thApr. 2001 at the Deutsche Sammlung für Mikroorganismen und Zellkulturen(DSMZ=German Collection of Microorganisms and Cell Cultures,Braunschweig, Germany) in accordance with the Budapest Treaty:

-   -   Escherichia coli Top10/pCR2.1mtrAint as DSM 14227,    -   Escherichia coli Top10/pCR2.1mtrBint as DSM 14228.

The present invention is explained in more detail in the following withthe aid of embodiment examples.

The isolation of plasmid DNA from Escherichia coli and all techniques ofrestriction, Klenow and alkaline phosphatase treatment were carried outby the method of Sambrook et al. (Molecular Cloning. A LaboratoryManual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., USA). Methods for transformation of Escherichia coli are alsodescribed in this handbook.

The composition of the usual nutrient media, such as LB or TY medium,can also be found in the handbook by Sambrook et al.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only, and are not intended to belimiting unless otherwise specified.

EXAMPLE 1

Preparation of a Genomic Cosmid Gene Library from C. glutamicum ATCC13032

Chromosomal DNA from C. glutamicum ATCC 13032 is isolated as describedby Tauch et al. (1995, Plasmid 33:168-179) and partly cleaved with therestriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany,Product Description Sau3AI, Code no. 27-0913-O₂). The DNA fragments aredephosphorylated with shrimp alkaline phosphatase (Roche MolecularBiochemicals, Mannheim, Germany, Product Description SAP, Code no.1758250). The DNA of the cosmid vector SuperCosl (Wahl et al. (1987),Proceedings of the National Academy of Sciences, USA 84:2160-2164),obtained from Stratagene (La Jolla, USA, Product Description SuperCoslCosmid Vector Kit, Code no. 251301) is cleaved with the restrictionenzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product DescriptionXbaI, Code no. 27-0948-02) and likewise dephosphorylated with shrimpalkaline phosphatase.

The cosmid DNA is then cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Codeno. 27-0868-04). The cosmid DNA treated in this manner is mixed with thetreated ATCC13032 DNA and the batch is treated with T4 DNA ligase(Amersham Pharmacia, Freiburg, Germany, Product DescriptionT4-DNA-Ligase, Code no.27-0870-04). The ligation mixture is then packedin phages with the aid of Gigapack II XL Packing Extract (Stratagene, LaJolla, USA, Product Description Gigapack II XL Packing Extract, Code no.200217).

For infection of the E. coli strain NM554 (Raleigh et al. 1988, NucleicAcids Res. 16:1563-1575) the cells are taken up in 10 mM MgSO₄ and mixedwith an aliquot of the phage suspension. The infection and titering ofthe cosmid library are carried out as described by Sambrook et al.(1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), thecells being plated out on LB agar (Lennox, 1955, Virology, 1:190)+100mg/l ampicillin. After incubation overnight at 37° C., recombinantindividual clones are selected.

EXAMPLE 2

Isolation and Sequencing of the mtrA and mtrB Genes

The cosmid DNA of an individual colony is isolated with the Qiaprep SpinMiniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) in accordancewith the manufacturer's instructions and partly cleaved with therestriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany,Product Description Sau3AI, Product No. 27-0913-O₂). The DNA fragmentsare dephosphorylated with shrimp alkaline phosphatase (Roche MolecularBiochemicals, Mannheim, Germany, Product Description SAP, Product No.1758250). After separation by gel electrophoresis, the cosmid fragmentsin the size range of 1500 to 2000 bp are isolated with the QiaExII GelExtraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

The DNA of the sequencing vector pZero-1, obtained from Invitrogen(Groningen, The Netherlands, Product Description Zero Background CloningKit, Product No. K2500-01) is cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI,Product No. 27-0868-04). The ligation of the cosmid fragments in thesequencing vector pzero-1 is carried out as described by Sambrook et al.(1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), theDNA mixture being incubated overnight with T4 ligase (Pharmacia Biotech,Freiburg, Germany). This ligation mixture is then electroporated (Tauchet al. 1994, FEMS Microbiol. Letters, 123:343-7) into the E. coli strainDH5αmcr (Grant, 1990, Proceedings of the National Academy of Sciences,U.S.A., 87:4645-4649). Letters, 123:343-7) and plated out on LB agar(Lennox, 1955, Virology, 1:190) with 50 mg/l zeocin.

The plasmid preparation of the recombinant clones is carried out with aBiorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). Thesequencing is carried out by the dideoxy chain-stopping method of Sangeret al. (1977, Proceedings of the National Academies of Sciences, U.S.A.,74:5463-5467) with modifications according to Zimmermann et al. (1990,Nucleic Acids Research, 18:1067). The “RR dRhodamin Terminator CycleSequencing Kit” from PE Applied Biosystems (Product No. 403044,Weiterstadt, Germany) was used. The separation by gel electrophoresisand analysis of the sequencing reaction are carried out in a“Rotiphoresis NF Acrylamide/Bisacrylamide” Gel (29:1) (Product No.A124.1, Roth, Karlsruhe, Germany) with the “ABI Prism 377” sequencerfrom PE Applied Biosystems (Weiterstadt, Germany).

The raw sequence data obtained are then processed using the Stadenprogram package (1986, Nucleic Acids Research, 14:217-231) version 97-0.The individual sequences of the pzerol derivatives are assembled to acontinuous contig.

The computer-assisted coding region analysis is prepared with the XNIPprogram (Staden, 1986, Nucleic Acids Research 14:217-231). Furtheranalyses are carried out with the “BLAST search program” (Altschul etal., 1997, Nucleic Acids Research, 25:3389-3402) against thenon-redundant databank of the “National Center for BiotechnologyInformation” (NCBI, Bethesda, Md., USA).

The resulting nucleotide sequence is shown in SEQ ID No. 1. Analysis ofthe nucleotide sequence shows two open reading frames of 681 base pairsand 1512 base pairs, which are called the mtrA gene and mtrB gene. ThemtrA gene codes for a polypeptide of 226 amino acids. The mtrB genecodes for a polypeptide of 503 amino acids.

EXAMPLE 3

Preparation of Integration Vectors for Integration Mutagenesis of themtrA and mtrB Genes

From the strain ATCC 13032, chromosomal DNA is isolated by the method ofEikmanns et al. (Microbiology 140: 1817-1828 (1994)). On the basis ofthe sequence of the mtrA and mtrB genes known for C. glutamicum fromExample 2, the following oligonucleotides are chosen for the polymerasechain reaction (SEQ ID NOS:4, 5, 6, and 7):

-   mtrA-int1:-   5′ TGA TGC TTC CAG GCA TGA AC 3′-   mtrA-int2:-   5′ GAT CGC CGA CTT CGA TGA TT 3′-   mtrb-int1:-   5′ ACG ATG ACC TGG TGG TCT CT 3′-   mtrB-int2:-   5′ GAC TCG AAT CGG TCC AAC TC 3′

The primers shown are synthesized by MWG Biotech (Ebersberg, Germany)and the PCR reaction is carried out by the standard PCR method of Inniset al. (PCR Protocols. A Guide to Methods and Applications, 1990,Academic Press) with the Taq-polymerase from Boehringer Mannheim(Germany, Product Description Taq DNA polymerase, Product No. 1 146165). With the aid of the polymerase chain reaction, the primers allowamplification of an internal fragment of the mtrA gene 237 bp in sizeand of an internal fragment of the mtrB gene 634 bp in size. Theproducts amplified in this way are tested electrophoretically in a 0.8%agarose gel.

The amplified DNA fragments are ligated with the TOPO TA Cloning Kitfrom Invitrogen Corporation (Carlsbad, Calif., USA; Catalogue NumberK4500-01) in each case in the vector pCR2.1-TOPO (Mead at al. (1991)Bio/Technology 9:657-663).

The E. coli strain TOP10 is then electroporated with the ligationbatches (Hanahan, In: DNA cloning. A Practical Approach. Vol. I,IRL-Press, Oxford, Washington D.C., USA, 1985). Selection forplasmid-carrying cells is made by plating out the transformation batchon LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual.2^(nd) Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), which has been supplemented with 50 mg/l kanamycin. PlasmidDNA is isolated from in each case a transformant with the aid of theQIAprep Spin Miniprep Kit from Qiagen and checked by restriction withthe restriction enzyme EcoRI and subsequent agarose gel electrophoresis(0.8%). The plasmids are called pCR2.1mtrAint and pCR2.1mtrBint and areshown in FIG. 1 and in FIG. 2.

EXAMPLE 4

Integration Mutagenesis of the mtrA Gene in the Strain DSM 5715

The vector pCR2.1mtrAint mentioned in Example 3 is electroporated by theelectroporation method of Tauch et al. (FEMS Microbiological Letters,123:343-347 (1994)) in Corynebacterium glutamicum DSM 5715. The strainDSM 5715 is an AEC-resistant lysine producer. The vector pCR2.1mtrAintcannot replicate independently in DSM5715 and is retained in the cellonly if it has integrated into the chromosome of DSM 5715. Selection ofclones with pCR2.1mtrAint integrated into the chromosome is carried outby plating out the electroporation batch on LB agar (Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2^(nd) Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), which has been supplementedwith 15 mg/l kanamycin.

For detection of the integration, the mtrAint fragment is labeled withthe Dig hybridization kit from Boehringer by the method of “The DIGSystem Users Guide for Filter Hybridization” of Boehringer Mannheim GmbH(Mannheim, Germany, 1993). Chromosomal DNA of a potential integrant isisolated by the method of Eikmanns et al. (Microbiology 140: 1817-1828(1994)) and in each case cleaved with the restriction enzymes KpnI,EcoRI and PstI. The fragments formed are separated by means of agarosegel electrophoresis and hybridized at 68° C. with the Dig hybridizationkit from Boehringer. The plasmid pCR2.1mtrAint mentioned in Example 3has been inserted into the chromosome of DSM5715 within the chromosomalmtrA gene. The strain is called DSM5715::pCR2.1mtrAint.

EXAMPLE 5

Integration Mutagenesis of the mtrB Gene in the Strain DSM

The vector pCR2.1mtrBint mentioned in Example 3 is electroporated by theelectroporation method of Tauch et al. (FEMS Microbiological Letters,123:343-347 (1994)) in Corynebacterium glutamicum DSM 5715. The strainDSM 5715 is an AEC-resistant lysine producer. The vector pCR2.1mtrBintcannot replicate independently in DSM5715 and is retained in the cellonly if it has integrated into the chromosome of DSM 5715. Selection ofclones with pCR2.1mtrBint integrated into the chromosome is carried outby plating out the electroporation batch on LB agar (Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2^(nd) Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), which has been supplementedwith 15 mg/l kanamycin.

For detection of the integration, the mtrBint fragment is labeled withthe Dig hybridization kit from Boehringer by the method of “The DIGSystem Users Guide for Filter Hybridization” of Boehringer Mannheim GmbH(Mannheim, Germany, 1993). Chromosomal DNA of a potential integrant isisolated by the method of Eikmanns et al. (Microbiology 140: 1817-1828(1994)) and in each case cleaved with the restriction enzymes KpnI,EcoRI and PstI. The fragments formed are separated by means of agarosegel electrophoresis and hybridized at 68° C. with the Dig hybridizationkit from Boehringer. The plasmid pCR2.1mtrBint mentioned in Example 3has been inserted into the chromosome of DSM5715 within the chromosomalmtrB gene. The strain is called DSM5715::pCR2.1mtrBint.

EXAMPLE 6

Preparation of Lysine

The C. glutamicum strain DSM5715::pCR2.1mtrBint obtained in Example 5 iscultured in a nutrient medium suitable for the production of lysine andthe lysine content in the culture supernatant is determined.

For this, the strain is first incubated on an agar plate with thecorresponding antibiotic (brain-heart agar with kanamycin 25 mg/l) for24 hours at 33° C. Starting from this agar plate culture, a precultureis seeded (10 ml medium in a 100 ml conical flask). The complete mediumCg III is used as the medium for the preculture. Medium Cg III NaCl 2.5g/l Bacto-Peptone  10 g/l Bacto-Yeast extract  10 g/l Glucose(autoclaved separately)  2% (w/v)The pH is brought to pH 7.4

Kanamycin (25 mg/l) is added to this. The preculture is incubated for 16hours at 33° C. at 240 rpm on a shaking machine. A main culture usseeded from this preculture such that the initial OD (660 nm) of themain culture is 0.1. Medium MM is used for the main culture. Medium MMCSL (corn steep liquor)   5 g/l MOPS (morpholinopropanesulfonic acid) 20 g/l Glucose (autoclaved separately)  50 g/l Salts: (NH₄)₂SO₄  25 g/lKH₂PO₄ 0.1 g/l MgSO₄ * 7 H₂O 1.0 g/l CaCl₂ * 2 H₂O  10 mg/l FeSO₄ * 7H₂O  10 mg/l MnSO₄ * H₂O 5.0 mg/l Biotin (sterile-filtered) 0.3 mg/lThiamine * HCl (sterile-filtered) 0.2 mg/l Leucine (sterile-filtered)0.1 g/l CaCO₃  25 g/l

The CSL, MOPS and the salt solution are brought to pH 7 with aqueousammonia and autoclaved. The sterile substrate and vitamin solutions arethen added, and the CaCO₃ autoclaved in the dry state is added.

Culturing is carried out in a 10 ml volume in 100 ml conical flasks withbaffles. Kanamycin (25 mg/l) was added. Culturing is carried out at 33°C. and 80% atmospheric humidity.

After 72 hours, the OD is determined at a measurement wavelength of 660nm with a Biomek 1000 (Beckmann Instruments GmbH, Munich). The amount oflysine formed is determined with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatographyand post-column derivation with ninhydrin detection.

The result of the experiment is shown in Table 1. TABLE 1 OD Lysine HClStrain (660 nm) g/l DSM5715 12.2 13.59 DSM5715::pCR2.1mtrBint 11.9 14.54

The abbreviations and designations used have the following meaning.

-   KMR: Kanamycin resistance gene-   KpnI: Cleavage site of the restriction enzyme KpnI-   EcoRI: Cleavage site of the restriction enzyme EcoRI-   PstI: Cleavage site of the restriction enzyme PstI-   mtrAint: Internal fragment of the mtrA gene-   mtrBint: Internal fragment of the mtrb gene-   ColE1: Replication origin of the plasmid ColE1

The present application claims priority to German Application No. DE10057802.0, which was filed on Nov. 11, 2001 and German Application No.DE 10125089.4, which was filed on may 23, 2001; the entire contents ofboth documents are incorporated herein by reference.

Obviously, numerous modifications and variations on the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1-39. (canceled)
 40. A Coryneform bacterium, which comprises anattenuated mtrA gene.
 41. The Coryneform bacterium of claim 40, whereinsaid mtrA gene comprises nucleotides 542-1219 of SEQ ID NO:1.
 42. TheCoryneform bacterium of claim 40, wherein said mtrA gene comprises anucleotide sequence that hybridizes under stringent conditions to apolynucleotide that is complimentary to nucleotides 542-1291 of SEQ IDNO:1, wherein said stringent conditions comprise washing in 5×SSC at atemperature of from 50 to 68° C.
 43. A Coryneform bacterium, whichcomprises an attenuated mtrB gene.
 44. The Coryneform bacterium of claim43, wherein said mtrB gene comprises nucleotides 1310 to 2818 of SEQ IDNO:1.
 45. The Coryneform bacterium of claim 43, wherein said mtrB genecomprises a nucleotide sequence that hybridizes under stringentconditions to a polynucleotide that is complimentary to nucleotides 1310to 2818 of SEQ ID NO: 1, wherein said stringent conditions comprisewashing in 5×SSC at a temperature of from 50 to 68° C.
 46. Escherichiacoli DSM
 14227. 47. Escherichia coli DSM
 14228. 48. A process forproducing L-amino acids comprising culturing a bacterial cell in amedium suitable for producing L-amino acids, wherein said bacterial cellcomprises an attenuated mtrA gene.
 49. The process of claim 48, whereinsaid bacterial cell is a Coryneform bacterium or Brevibacterium.
 50. Theprocess of claim 49, wherein said bacterial cell is selected from thegroup consisting of Coryneform glutamicum, Corynebacteriumacetoglutamicum, Corynebacterium acetoacidophilum, Corynebacteriummelassecola, Corynebacterium thermoaminogenes, Brevibacterium flavum,Brevibacterium lactofermentum, and Brevibacterium divaricatum.
 51. Theprocess of claim 49, wherein said mtrA gene comprises the nucleotides542 to 1219 of SEQ ID NO:1.
 52. The process of claim 49, wherein saidmtrA gene comprises a nucleotide sequence that hybridizes understringent conditions to a polynucleotide that is complimentary tonucleotides 542-1291 of SEQ ID NO: 1, wherein said stringent conditionscomprise washing in 5×SSC at a temperature of from 50 to 68° C.
 53. Theprocess of claim 49, wherein said L-amino acid is L-lysine.
 54. Theprocess of claim 49, wherein said bacteria further comprises anattenuated mtrB gene.
 55. The process of claim 54, wherein said mtrBgene comprises nucleotides 1310 to 2818 of SEQ ID NO:1.
 56. The processof claim 54, wherein said mtrB gene comprises a nucleotide sequence thathybridizes under stringent conditions to a polynucleotide that iscomplimentary to nucleotides 1310 to 2818 of SEQ ID NO: 1, wherein saidstringent conditions comprise washing in 5×SSC at a temperature of from50 to 68° C.
 57. The process of claim 49, wherein said bacteria furthercomprises at least one gene whose expression is enhanced, wherein saidgene is selected from the group consisting of dapA, gap, tpl, pgk, zwf,pyc, mqo, lysC, lysE, hom, ilvA, ilvBN, ilvD and zwa
 1. 58. The processof claim 49, wherein said bacteria further comprises at least one genewhose expression is attenuated, wherein said gene is selected from thegroup consisting of pck, pgi, poxB, and zwa2.
 59. A process forproducing L-amino acids comprising culturing a bacterial cell in amedium suitable for producing L-amino acids, wherein said bacterial cellcomprises an attenuated mtrB gene.
 60. The process of claim 59, whereinsaid bacterial cell is a Coryneform bacterium or Brevibacterium.
 61. Theprocess of claim 60, wherein said bacterial cell is selected from thegroup consisting of Coryneform glutamicum, Corynebacteriumacetoglutamicum, Corynebacterium acetoacidophilum, Corynebacteriummelassecola, Corynebacterium thermoaminogenes, Brevibacterium flavum,Brevibacterium lactofermentum, and Brevibacterium divaricatum.
 62. Theprocess of claim 59, wherein said mtrB gene comprises the nucleotides1310 to 2818 of SEQ ID NO:1.
 63. The process of claim 59, wherein saidmtrB gene comprises a nucleotide sequence that hybridizes understringent conditions to a polynucleotide that is complimentary tonucleotides 1310 to 2818 of SEQ ID NO:1, wherein said stringentconditions comprise washing in 5×SSC at a temperature of from 50 to 68°C.
 64. The process of claim 59, wherein said L-amino acid is L-lysine.65. The process of claim 59, wherein said bacteria further comprises atleast one gene whose expression is enhanced, wherein said gene isselected from the group consisting of dapA, gap, tpl, pgk, zwf, pyc,mqo, lysC, lysE, hom, ilvA, ilvBN, ilvD and zwa
 1. 66. The process ofclaim 59, wherein said bacteria further comprises at least one genewhose expression is attenuated, wherein said gene is selected from thegroup consisting ofpck, pgi, poxB, and zwa2. 67-78. (canceled)